Pesticides have enjoyed widespread use in commercial agriculture and have enabled an enormous increase in crop yields and product quality. Pesticides are also routinely used to control various insects, for example, flies or mosquitoes. Often, pest populations pose a nuisance or health hazard to humans or livestock. There is, however, an increasing awareness of environmental risks associated with the use of certain synthetic pesticides, including concern over the bioaccumulation of pesticides in the food chain or their detrimental effects on non-target organisms. Biological pesticides and, especially, natural biopesticides have, therefore, been of considerable interest to those seeking environmentally acceptable means of pest control.
The microorganism Bacillus thuringiensis (B. thuringiensis) has long been known to be useful in the control of insect pests. The sporulating B. thuringiensis cell produces a class of compounds, formerly regarded as a single δ-endotoxin, but now understood to comprise several distinct toxin proteins, which are concentrated in a crystalline protein inclusion body found in the endospore. Upon ingestion of the inclusion body by a susceptible insect larva and proteolysis in the insect gut, the endotoxin proteins are converted into active compounds which destroy the gut epithelium and, ultimately, the pest itself.
B. thuringiensis δ-endotoxins have accordingly been found to be useful as pesticides when applied in the form of lysates or other fermentation extracts of cultures of the microorganism.
There are several Bacillus thuringiensis strains that are widely used as biopesticides in the forestry, agricultural, and public health areas. Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai produce delta-endotoxins specific for Lepidoptera. A delta-endotoxin specific for Coleoptera is produced by Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Pat. No. 4,766,203). Furthermore, Bacillus thuringiensis subsp. israelensis produces delta-endotoxins specific for Diptera (Goldberg, 1979, U.S. Pat. No. 4,166,112).
Other Bacillus thuringiensis strains specific for dipteran pests have also been described. A Bacillus thuringiensis isolate has been disclosed which is toxic to Diptera and Lepidoptera (Hodgman et al., 1993, FEMS Microbiology Letters 114:17-22). Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) of the purified crystal delta-endotoxin from this isolate revealed three protein species which are related to Cry1Ab, Cry1B, and Cry2A toxins. There has also been disclosed a Bacillus thuringiensis isolate which produces a dipteran-active crystal comprised of proteins with molecular weights of 140, 122, 76, 72, and 38 kDa (Payne, 1994, U.S. Pat. No. 5,275,815). EP 480,762 discloses five B.t. strains which are each active against dipteran pests; each also has a unique crystal delta-endotoxin pattern.
Several Bacillus thuringiensis strains have been described which have pesticidal activity against pests other than Lepidoptera, Coleoptera, and Diptera. Five Bacillus thuringiensis strains have been disclosed which produce delta-endotoxins that are toxic against nematodes (Edwards, Payne, and Soares, 1988, Eur. Pat. Appl. No. 0 303 426 B1). There has also been disclosed a Bacillus thuringiensis strain, PS81F, which can be used to treat humans and animals hosting parasitic protozoans (Thompson and Gaertner, 1991, Eur. Pat. Appl. No. 0 461 799 A2). Several Bacillus thuringiensis isolates have also been disclosed with activity against acaride pests. These isolates produce crystals comprised of proteins with molecular weights in the range of 35 kDa to 155 kDa (Payne, Cannon, and Bagley, 1992, PCT Application No. WO 92/19106). There have also been disclosed Bacillus thuringiensis strains with activity against pests of the order Hymenoptera (Payne, Kennedy, Randall, Meier, and Uick, 1992, Eur. Pat. Appl. No. 0 516 306 A2); Hemiptera (Payne and Cannon, 1993, U.S. Pat. No. 5,262,159); fluke pests (Hickle, Sick, Schwab, Narva, and Payne, 1993, U.S. Pat. No. 5,262,399); and pests of the order Phthiraptera (Payne and Hickle, 1993, U.S. Pat. No. 5,273,746). Furthermore, another strain of Bacillus thuringiensis subsp. kurstaki, WB3S-16, isolated from Australian sheep wool clippings, has been disclosed that is toxic to the biting louse Damalinia ovis, a Phthiraptera pest (Drummond, Miller, and Pinnock, 1992, J. Invert. Path. 60:102-103).
The delta-endotoxins are encoded by cry (crystal protein) genes which are generally located on plasmids. The cry genes have been divided into more than 50 classes and several subclasses based on relative amino acid homology and pesticidal specificity. The major classes are Lepidoptera-specific (cry1); Lepidoptera- and Diptera-specific (cry2); Coleoptera-specific (cry3); Diptera-specific (cry4) (Hofte and Whiteley, 1989, Microbiological Reviews 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cry5 genes by Tailor et al., 1992, Molecular Microbiology 6:1211-1217); and Nematode-specific (referred to as cry5 and cry6 genes by Feitelson et al., 1992, Bic)/Technology 10:271-275). A current list of cry toxins can be found, for example, in Crickmore, N., Zeigler, D. R., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J, Bravo, A. and Dean, D. H. “Bacillus thuringiensis toxin nomenclature” (2009); Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins, N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean. Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813.
Delta-endotoxins may or may not be in crystal form, and have been produced by recombinant DNA methods.
There is a continued need for the identification of novel B. thuringiensis strains which display a broader or different spectrum of, or an increased level of pesticide activity.